1. Field of the Invention
The present invention relates generally to the field of protein therapeutics. More particularly, it concerns improved methods and compositions for producing human arginases modified for long stability and persistence in humans.
2. Description of Related Art
It has been recognized for over 50 years that certain tumor cells have a high demand for L-Arginine and are killed under conditions of L-Arginine depletion (Wheatley and Campbell, 2002). In human cells L-Arginine is synthesized in two steps: first argininosuccinate synthetase (ASS) converts L-Citrulline and L-Aspartate to argininosuccinate, followed by conversion of argininosuccinate to L-Arginine and fumarate by argininosuccinate lyase. L-Citrulline itself is synthesized from L-Ornithine and carbamoyl phosphate by the enzyme ornithine transcarbamylase (OTC). A large number of hepatocellular carcinomas, melanomas, and, as discovered recently, renal cell carcinomas (Ensor et al., 2002; Feun et al., 2007; Yoon et al., 2007) do not express ASS and thus are sensitive to L-Arginine depletion. The molecular basis for the lack of ASS expression appears to be diverse and includes aberrant gene regulation and splicing defects. Whereas non-malignant cells enter into quiescence (G0) when depleted of L-Arginine and thus remain viable for several weeks, tumor cells have cell cycle defects that lead to the re-initiation of DNA synthesis even though protein synthesis is inhibited, in turn resulting in major imbalances and rapid cell death (Shen et al., 2006; Scott et al., 2000). The selective toxicity of L-Arginine depletion for HCC, melanoma and other ASS-deficient cancer cells has been extensively demonstrated in vitro, in xenograft animal models and in clinical trials I Shen et al., 2006; Ensor et al., 2002; Feun et al., 2007; Izzo et al., 2004). Recently Cheng et al. (2007) demonstrated that many HCC cells are also deficient in ornithine transcarbamylase expression and thus, they are also susceptible to enzymatic L-Arginine depletion.
There is interest in the use of L-Arginine hydrolytic enzymes for cancer therapy. Two L-Arginine degrading enzymes have been studied for cancer therapy: bacterial arginine deiminase and human Arginase. Unfortunately, both of these enzymes display significant shortcomings that present major impediments to clinical use (immunogenicity and low catalytic activity in serum, respectively). Thus, there is a need to develop improved compositions and methods for L-Arginine depletion cancer therapy, especially to develop therapeutics for L-Arginine depletion therapy that display favorable pharmacokinetics in humans including long persistence in circulation